In order to obtain a bacterial strain which produces an amino acid efficiently, it is important to know properties of genes relating to the biosynthesis of the amino acid in the bacterium and their style for controlling expression and activity and to carry out rational breeding based thereon.
One of the important methods for understanding the functions of genes relating to the amino acid production is a genetic method, for example, in which a relationship between increase or decrease in amino acid productivity and gene mutation is clarified.
Breeding of amino acid-producing microorganisms is mainly carried out by endowing resistance to drugs such as amino acid analogues and the like, but in many cases, it is not clear which gene provides the productivity improvement by its mutation.
NADPH is necessary as a coenzyme at reduction reaction in the amino acid biosynthesis in many microorganisms. For example, 4 molecules of NADPH are necessary for the biosynthesis of 1 molecule of L-lysine. In the same manner, 3 molecules of NADPH are necessary for 1 molecule of threonine, and 5 molecules of NADPH are necessary for 1 molecule of isoleucine. Thus, two or more molecules of NADPH are necessary for the biosynthesis of 1 molecule of most amino acids. Accordingly, supply of NADPH is an important subject in producing these amino acids using microorganisms.
In many microorganisms, NADPH-supplying enzymes are limited. It is considered that the enzymes which can supply NADPH on the main pathways of sugar metabolism of the microorganisms are mainly G6PD [EC 1.1.1.49] and 6-phosphogluconate dehydrogenase [EC 1.1.1.4] in the pentose phosphate pathway (HMP) and isocitrate dehydrogenase [EC-1.1.1.41] in the TCA pathway.
Particularly, G6PD, which is the first enzyme of HMP and is also the parting point-enzyme from the Embden-Meyerhof pathway (EMP), is considered to be a very important enzyme for the production of various amino acids by bacteria belonging to the genus Escherichia and the genus Corynebacterium , and various analyses have been carried out mainly on its various biochemical properties. For example, G6PD of bacteria belonging to the genus Corynebacterium is described in Journal of Bacteriology, 98, 1151 (1969), Agricultural and Biological Chemistry, 51, 101 (1987) and Japanese Published Unexamined Patent Application No. 224661/97, but the investigation for productivity improvement of amino acids using the enzyme has not been reported.
Also, the nucleotide sequence of G6PD of bacteria such as Escherichia coli and Corynebacterium glutamicum, the nucleotide sequence of the gene has been found (Journal of Bacteriology, 173, 968 (1991) and Japanese Published Unexamined Patent Application No. 224661/97), but the investigation for productivity improvement of amino acids using the gene has not been reported.